Research ArticleEndometrial Receptivity Profile in Patients with PrematureProgesterone Elevation on the Day of hCG Administration
Delphine Haouzi,1,2,3 Laurence Bissonnette,1,2,3,4 Anna Gala,5
Said Assou,1,2,3 Frida Entezami,6 Hélène Perrochia,7 Hervé Dechaud,1,2,3,5
Jean-Noel Hugues,8 and Samir Hamamah1,2,3,5
1 CHU Montpellier, Institut de Recherche en Biotherapie, Hopital Saint-Eloi, 34295 Montpellier, France2 INSERM U1040, Hopital Saint-Eloi, 34295 Montpellier, France3 Universite Montpellier 1, UFR de Medecine, Equipe “Developpement Embryonnaire Precoce et CellulesSouches Embryonnaires Humaines”, 34000 Montpellier, France
4OVO Fertility, 8000 Boulevard Decarie No. 100, Montreal, QC, Canada H4P 2S45 CHUMontpellier, ART/PGD Division, Departement de Biologie de la Reproduction,Hopital Arnaud de Villeneuve, 34295 Montpellier, France
6 Laboratoire Dynabio, Polyclinique du Cotentin, 50120 Equeurdreville, France7 CHUMontpellier, Hopital Gui de Chauliac, Service Anatomie Cytologie Pathologiques, 34295 Montpellier, France8 CHU Leonard de Vinci-Universite Paris XIII, Service de Medecine de la Reproduction, Hopital Jean Verdier, 93143 Bondy, France
Correspondence should be addressed to Samir Hamamah; [email protected]
Received 16 December 2013; Revised 3 April 2014; Accepted 3 April 2014; Published 28 April 2014
The impact of a premature elevation of serum progesterone level, the day of hCG administration in patients under controlledovarian stimulation during IVF procedure, on human endometrial receptivity is still debated. In the present study, we investigatedthe endometrial gene expression profile shifts during the prereceptive and receptive secretory stage in patients with normal andelevated serum progesterone level on the day of hCG administration in fifteen patients under stimulated cycles. Then, specificbiomarkers of endometrial receptivity in these two groups of patients were tested. Endometrial biopsies were performed on oocyteretrieval day and on day 3 of embryo transfer, respectively, for each patient. Samples were analysed using DNA microarrays andqRT-PCR.The endometrial gene expression shift from the prereceptive to the receptive stagewas altered in patients with high serumprogesterone level (>1.5 ng/mL) on hCG day, suggesting accelerated endometrial maturation during the periovulation period.Thiswas confirmed by the functional annotation of the differentially expressed genes as it showed downregulation of cell cycle-relatedgenes. Conversely, the profile of endometrial receptivity was comparable in both groups. Premature progesterone rise alters theendometrial gene expression shift between the prereceptive and the receptive stage but does not affect endometrial receptivity.
1. Introduction
The impact of premature serum progesterone elevation atthe end of the follicular phase under controlled ovarianstimulation (COS) cycle for in vitro fertilization (IVF) isstill debated. While several studies reported lower pregnancyrates in patients with high progesterone concentration on theday of human chorionic gonadotropin (hCG) administration[1–9], one found a favourable effect on pregnancy outcome
[10] and others failed to demonstrate any association [11–21]. Although the mechanism by which premature serumprogesterone elevation might alter the embryo transfer out-come is still unclear, there are accumulated data suggesting anegative impact on endometrium [22, 23]. Elevated proges-terone levels might induce premature endometrial matura-tion and, as a consequence, earlier opening of the implanta-tion window that leads to asynchronization of the crosstalkbetween embryo and endometrium. Accelerated endometrial
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 951937, 10 pageshttp://dx.doi.org/10.1155/2014/951937
maturation following COS has been clearly demonstrated byhistological dating on the day of oocyte retrieval [24–27], butthis is not the case during the implantation window [22].Therefore, to what extent endometrial receptivity is impairedin patients with high serum progesterone level is question-able.
In addition, very few studies have assessed the impactof serum progesterone elevation on the endometrial geneexpression profile during the implantation window [22, 23](Table 1). By comparing the endometrial gene expressionprofiles during the implantation window in patients withhigh and normal serum progesterone level on the day ofhCG administration, these authors found significant differ-ences in the expression of genes that play a crucial role inendometrial function. Although some of these genes wererelated to endometrial receptivity, no clear assessment of theendometrial status during the implantation window in thepatients with high serum progesterone level on hCG day wascarried out.
The aim of the study was (i) to compare individuallythe endometrial gene expression shift between the prerecep-tive and receptive secretory stages in patients with normal(<1.5 ng/mL) and high (>1.5 ng/mL) serum progesteronelevels on the day of hCG administration and, then, (ii) to testbiomarkers of endometrial receptivity in these two groups ofpatients.
2. Materials and Methods
2.1. Patients’ Characteristics and Endometrial Biopsies. Thestudy population included 15 patients (age 31 years ±3) whowere referred for intracytoplasmic sperm injection (ICSI) formale infertility and were recruited after written informedconsent.This project was approved by the Ethical Committeeof the Institut de Recherche en Biotherapie. All patients hadnormal serum FSH, LH, and estradiol on day 3 of COS undereither GnRH agonist long or antagonist protocols, as well ason the day of hCG administration (Table 2). An endometrialbiopsy was obtained on the day of oocyte collection (hCG+2)and another one during embryo transfer (hCG+5), respec-tively. Endometrial biopsies (𝑛 = 30) were washed and frozenindividually at −80∘C prior to total RNA extraction with theRNeasy Mini Kit (Qiagen, Valencia, CA, USA).
2.2. Progesterone Measurement. Serum progesterone wasmeasured on the day of hCG administration by usingan automated Cobas e411 instrument (Roche Diagnostics).Intra-assays and interassay coefficients of variation (CV)were<2.7% and <9.1%, respectively.
2.3. Microarray Hybridization. Total RNA (100 ng) was usedto prepare twice amplified and labelled cRNA for hybridiza-tion with HG-U133 plus 2.0 arrays (Affymetrix, UK) asdescribed in [29]. Each endometrial sample was processedindividually on a separate DNA microarray chip.
2.4. Data Processing and Microarray Data Analysis. ScannedGeneChip images were processed using the AffymetrixGCOS 1.4 software to obtain the intensity value signal
and the absent/present detection call for each probe setusing the default analysis settings and global scaling as firstnormalization method. Probe intensities were derived usingthe MAS5.0 algorithm.
Patients (𝑛 = 15) were divided into two groups accordingto their serum progesterone concentration ([P]) on the dayof hCG administration: <1.5 ng/mL (normal [P] group, 𝑛 = 7patients) and >1.5 ng/mL (high [P] group, 𝑛 = 8 patients)(Table 2). The number of patients under GnRH long agonistprotocol was similar in each group (2 per group). To comparethe endometrial gene expression profile shift between hCG+2and hCG+5 samples in the two groups of patients, a probe setselection using the detection call (present in all samples of theselected group) and a coefficient of variation ≥40% betweenendometrial samples were first carried out. Then, the signifi-cant analysis of microarrays (SAM; Stanford University) [30]was used to identify genes thatwere significantly differentiallyexpressed between the hCG+2 and hCG+5 endometriumsamples (paired-sample analysis) from the normal and high[P] groups. The lists of identified genes (fold change, FC >2; false discovery rate, FDR < 5%) were submitted to Inge-nuity (http://www.ingenuity.com) to identify the biologicalpathways/functions that were specific of the high serum [P]group. Unsupervised hierarchical clustering analyses wereperformed with the Cluster and TreeView software packages.
2.5. Quantitative RT-PCR Analyses. To assess biomarkers ofendometrial receptivity, RNA (0.5𝜇g) of receptive endome-trium samples from patients with normal (hCG+5, 𝑛 = 3)and high [P] (hCG+5, 𝑛 = 3) on the day of hCG admin-istrationwas used for reverse transcription-quantitative poly-merase chain reaction (RT-qPCR) according to the manufac-turer’s recommendations (Applied Biosystems, Villebon surYvette, France). To validate some microarray data comparingthe endometrial gene expression shift between prereceptiveand receptive secretory stages, RNA (0.5 𝜇g) of prereceptivesamples from patients with normal (hCG+2, 𝑛 = 3) andhigh [P] (hCG+2, 𝑛 = 3) was also used. For qPCR, 2𝜇L(of a 1 : 5 dilution) of the first strand DNA was added to a10 𝜇L reaction mixture containing 0.25 𝜇M of each primerand 5 𝜇L of 2X LightCycler 480 SYBR Green I Master mix(Roche, Mannheim, Germany). DNA was amplified during45 cycles with annealing temperature at 63∘C using the LightCycler 480 detection system (Roche). The sample valueswere normalized toPGK1 (phosphoglycerate kinase 1) expres-sion using the following formula: 𝐸ΔCttested primer/𝐸
ΔCt𝑃𝐺𝐾1
(𝐸 =10−1/slope), ΔCt = Ct control—Ct unknown, where 𝐸 corre-
sponds to the efficiency of the PCR reaction. The 𝐸 valuewas obtained by a standard curve that varies in function ofthe primers used. One receptive endometrium sample froma patient with normal serum [P] was used as control. Eachsample was analysed in duplicate and multiple water blankswere included.
2.6. Statistical Analyses. Statistical analyses of the clinical andqRT-PCR data were performed using the GraphPad InStat
BioMed Research International 3
Table1:Designof
threem
icroarray-basedstu
dies
thatinvestigated
theimpactof
high
serum
progesterone
levelonthee
ndom
etria
lgenee
xpressionprofi
le.
Stud
yStud
ypo
pulatio
nNum
bero
fpatie
nts
Num
bero
fsamples
Samplingtim
e[P]c
utt-o
ff(ng/mL)
Com
paredsamples
Fold
change
Num
bero
fgenes
Up
Dow
n
[22]
Oocyted
onors
1212
hCG+7
1.5hC
G+7,[P]>1.5
versus
hCG+7,[P]<1.5
≥2
6476
(𝑛=6)
(𝑛=6)
[23]
Patie
ntsu
ndergoingIV
Fdu
eto
tubalorm
aleinfertility
1010
hCG+8
1.2,1.9
hCG+8
,[P]<1.2
versus
hCG+8
,[P]>1.9
≥2
139
(𝑛=5)
((𝑛=5)
[27]
Not
specified
1414
hCG+2
1,1.5
hCG+2
,[P]≤0.9versus
hCG+2
,1>[P]>
1.5
≥1.4
5∗23∗
(𝑛=3)
((𝑛=6)
hCG+2
,1>[P]>
1.5versus
hCG+2
,[P]>1.5
607∗
212∗
(𝑛=6)
((𝑛=5)
Thisstu
dyNormoo
vulatorywom
enreferred
forICS
I15
30hC
G+2
,hCG
+51.5
hCG+2
[P]<
1.5versus
hCG+5
[P]<
1.5
>2
877
600
(𝑛=7)
((𝑛=7)
hCG+2
[P]>
1.5versus
hCG+5
[P]>
1.5123
110(𝑛=8)
((𝑛=8)
∗
Dataa
reexpressedin
numbero
fprobe
sets;𝑛:num
bero
fsam
ples;ICS
I:intracytop
lasm
icsperm
injection.
4 BioMed Research International
Number of genes
Up Down
877 600
123 110
hCG+2 versus hCG+5
[P] < 1.5ng/mL
[P] > 1.5ng/mL
(a)
1456 21221
[P] < 1.5ng/mL [P] > 1.5ng/mL
(b)
Figure 1: (a) Number of genes that were up- or downregulated in the normal and high [P] groups. (b) Venn diagram of the transcripts thatwere differentially expressed in the prereceptive and the receptive endometrial samples from patients with normal or high serum [P].
Table 2: Patients’ clinical characteristics on the day of hCG administration and pregnancy outcome.
[P] <1.5 ng/mL (𝑛 = 7) [P] >1.5 ng/mL (𝑛 = 8) 𝑃 valueAge (years) 31 ± 4.9 30.1 ± 2.7 NS[P] (ng/mL) 0.95 ± 0.24 2.6 ± 0.91 <0.001E2 (pg/mL) 2509 ± 1357 2682 ± 2098 NSLH (mIU/mL) 1.19 ± 0.19 1.25 ± 0.96 NSTotal FSH dose (IU) 1830 ± 414 1962 ± 262 NSNumber of retrieved oocytes 11.6 ± 5.2 15.5 ± 7.6 NSPregnancy (%)∗ 28.6 12.5 NSData are the mean ± SEM. NS: nonsignificant. ∗According to the serum 𝛽-human chorionic gonadotrophin measured 16 days after embryo transfer.
3 software. Differences between groups were consideredsignificant when Student’s 𝑡-test gave a 𝑃 value < 0.05.
3. Results
3.1. COS Parameters and Embryo Transfer Outcome. COSparameters and embryo transfer outcome were not signifi-cantly different between groups but for the serum [P] on theday of hCG administration (Table 2).
3.2. Gene Expression Profile Shifts between the Prereceptive andReceptive Secretory Stage in the Normal and High Serum [P]Groups. Using the detection call and the variation coefficientfor a first selection of genes expressed in the hCG+2 andhCG+5 endometrium samples, 6084 and 6130 genes wereidentified in the normal and high [P] groups, respectively.Then, SAM analysis of these microarray data identified 1477and 233 genes that were differentially modulated betweenthe two endometrial stages in the normal and high [P]group, respectively. The proportion of upregulated (59% inthe normal serum group and 53% in the high serum [P]group) and downregulated genes (41% in the normal serumgroup and 47% in the high serum [P] group) was similar inthe two groups of patients (Figure 1(a)). However, the foldchanges weremore important in the normal group [P] (−48.9≤ fold change ≤ 79.9) than in the high [P] group (−5.9 ≤ foldchange ≤ 40.4).
3.3. Cell Cycle-Related Genes Are Downregulated in the HighSerum [P] Group. To identify endometrial genes that werespecifically modulated in the high serum [P] group betweenthe prereceptive (hCG+2) and receptive (hCG+5) stage, wecrossintersected the lists of genes that were differentiallyexpressed between the two stages in the endometriumsamples from the high (𝑛 = 233 genes) and normal(𝑛 = 1477 genes) [P] groups (Figure 1(b)). We identified212 genes that were exclusively modulated in the high [P]group. Among them, more than 50 genes were involvedin the cell cycle [𝑃 value = 2.22 E –11 – 2.41 E −02],including several members of the cell division cycle fam-ily (CDC20, CDC25C, CDCA1, CDCA2, CDCA5, CDCA8),cyclins (CCNB1, CCNB2), and kinesins (KIF4A, KIF11, KIF15,KIF23) (Figure 2 and Table S1 for the complete list) (seeTable S1 in the Supplementary Material available onlineat http://dx.doi.org/10.1155/2014/951937); 75% of these geneswere downregulated. Some of these genes (CDCA2, CDCA8,FOXOA1, and TGFB2) have been validated by qRT-PCR(Figure 3). In addition, we identified 21 genes that werecommon to both groups (Table 3).
3.4. Endometrial Receptivity in Patients with High SerumProgesterone Level. To assess whether endometrial recep-tivity was affected in the high serum [P] group, we usedthe endometrial receptivity predictor list (54 genes) that wepreviously described [28] for unsupervised clustering of the
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UV-inducedDNA damage
CHK2
PLK
SLK CDC25
CDC25P
CDC2
CyclinB
P
PCHK1
RPRM
EG5PEG5
Separation of centromeresto opposite poles of the cell
APC
CyclinB
APC
FZR1
PLK
CDC20PRC1
TGF𝛽
(a) Mitotic entry
Cyclin BKIF4A
KIF11 Mpf
PBK
PRC1
DLGAP5
CCNB2
CDC25C
G0S2
E2F7CDC20BUB1B
NUF2
NDC80
SPC25CCNB1
BUB1 AURKA
NF𝜅B (complex)
FOXO1∗
NEK2∗
DEPDC1∗
(b) Network around the cell cycle
Figure 2: Signalling pathways that were altered in the high serum [P] group. The Ingenuity Pathway software was used to identify thefunctional pathways associated with genes that were differentially expressed between the prereceptive and receptive endometrial stages inthe high [P] group. The majority of genes related to mitotic entry (a) and cell cycle (b) were downregulated. In this network, edge types areindicatives: a plain line indicates direct interactions, a dashed line indicates indirect interactions, a line without arrowhead indicates bindingonly, a line finishing with a vertical line indicates inhibition, and a line with an arrowhead indicates “acts on”. Green, downregulated; red,upregulated.
Normal P High P
mRN
As (
a.u.)
0
2
4
6
CDCA2
mRN
As (
a.u.)
0
0.5
1
1.5
2
CDCA8
mRN
As (
a.u.) TGFB2
0
1
2
3
mRN
As (
a.u.)
0
0.5
1
1.5
FOXOA1
Normal P High P
Normal P High PNormal P High P
P = 0.032P = 0.043
P = 0.022P = 0.66
Figure 3: Validation by quantitative RT-PCR analysis of genes related to the cell cycle functionmodulated in the high [P] group. RNA isolatedfrom hCG+2 and hCG+5 endometrial samples of normal and high serum [P] patients (𝑛 = 3/each group) was used. Data are the mean ±SEM. NS, nonsignificant.
6 BioMed Research International
Table 3: List of genes shared by the normal serum [P] and highserum [P] groups.
endometrial gene expression profiles at hCG+2 (prerecep-tive) and hCG+5 (receptive stage). A clear segregation of thehCG+2 and hCG+5 endometrium samples independently ofserum [P] was observed, suggesting a similar transcriptomicshift of endometrial receptivity biomarkers in the two groups(Figure 4).
The most overexpressed predictors (𝑛 = 13) of theendometrial receptivity were selected for validation by qRT-PCR. No significant difference in the two groups of patients(normal versus high [P]) was observed, except for CD68 andKRT80 (Figure 5).
4. Discussion
Our data indicate that premature progesterone elevationalters the endometrial gene expression shift from the pre-receptive to the receptive stage. Indeed, the transcriptomicendometrial gene expression shift in the normal serum [P]group was comparable to the one previously described forpatients in COS protocols [29]. In the high serum [P] group,this transcriptomic shift was reduced with only 233 genesdifferentially expressed between hCG+2 and hCG+5. Thisfinding suggests that in the high [P] group endometrial mat-uration is accelerated during the early secretory phase. Thishypothesis was confirmed by the functional annotation anal-ysis as it revealed that many of the downregulated genes areinvolved in cell cycle functions. Previous studies [24, 27] alsoshowed an advanced endometrial maturation (2 to 4 days)based on histological dating on the day of oocyte retrieval
hCG+2, normal [P]hCG+5, normal [P]
hCG+2, high [P]hCG+5, high [P]
Figure 4: Unsupervised hierarchical clustering of the prereceptiveand receptive endometrium samples using the previously described[28] predictor list. Comparison of the gene expression profiles athCG+2 and hCG+5 in the normal and high serum [P] groupsrevealed similar transcriptomic profiles. Green, downregulated; red,upregulated.
(hCG+2). However, this was irrespective of the serum [P] onthe day of hCG administration. Furthermore, it is well knownthat progesterone might inhibit the normal endometrialproliferation [31]. More precisely, influence of progesteroneon cell proliferation appears dose dependent. Consequently,this can explain the downregulation of genes related to the cellcycle functions in patients with high serum [P]. This findingwas in conjunction with functional annotations reported byLabarta et al. [22] revealing alteration of the cellular growthand proliferation in patients with high serum progesteronelevel under COS. Simultaneous to its critical role in thecontrol of the proliferation status, progesterone is necessaryto the acquisition of the decidualization morphotype, a keyevent for acquisition of the receptive endometrial status and,therefore, successful implantation.
However, alteration of the endometrial transcriptomeshift in patients with high serum [P] did not seem to affectendometrial receptivity. Indeed, we observe by quantitativeRT-PCR analysis that most endometrial receptivity biomark-ers (genes that are upregulated during the implantation win-dow) display similar or higher expression levels in patientswith high [P] in comparison to women with normal serum[P]. These biomarkers were selected based on our previousstudy that identified new biomarkers of human endometrialreceptivity by comparing the endometrial gene expressionprofile shift during the prereceptive and receptive secretorystages in the same patients during a natural cycle [28]. Two ofthem (IL15 and SPP1) were identified by all six transcriptomicstudies that compared the same endometrial samples fromwomen in natural cycles [32].
Labarta et al. [22] examined 25 windows of implantationgenes that are strongly related to endometrium receptivenessand embryo implantation and were previously described in[33]. However, only 8 of these genes were referred in theirgenomic diagnostic tool as specific to endometrial receptivity[34]. Based on the expression of these eight biomarkers,they concluded that endometrial receptivity was affected inpatients with high serum [P] [22]. However, by thoroughlyanalysing their data, we think that the expression of these
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mRN
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a.u.)
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a.u.)
mRN
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a.u.)
mRN
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a.u.)
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a.u.)
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As (
a.u.)
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a.u.)
BCL2L10
NLF2
SORCS1
MFAP5
LAMB3
FST
ANGPTL1
PROK1
TRPC4
IL15
Normal P High PNormal P High P
P = 0.38
P = 0.94
P = 0.46 P = 0.87
P = 0.23
P = 0.67
P = 0.07 P = 0.6
P = 0.69
P = 0.06
(a)
Figure 5: Continued.
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KRT80
mRN
As (
a.u.)
Normal P High P
Normal P High PNormal P High P
∗P = 0.01
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SPP1
mRN
As (
a.u.)
P = 0.65
0
50
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150
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250
CD68
mRN
As (
a.u.)
∗P = 0.01
(b)
Figure 5: Validation by quantitative RT-PCR analysis of several biomarkers of endometrial receptivity using RNA isolated from hCG+5endometrial samples of normal andhigh serum [P] patients (𝑛 = 3/each group).Data are themean± SEM. ∗𝑃 value< 0.05;NS, nonsignificant.
eight biomarkers was increased in both fertile women withnatural cycles and in patients with high serum [P], albeit to alesser extent, thus strongly suggesting that endometrial recep-tivity was not affected in patients with high serum [P]. Inaddition, using the approach described by Labarta et al. [22],we found no significant differences in the gene expressionprofiles of receptive endometrium samples frompatients withhigh or normal serum [P], whatever the [P] threshold on theday of hCG administration (data not shown), thus stronglysuggesting again that endometrial receptivity is similar inpatients with normal and high [P].
Therefore, our findings point to an abnormally acceler-ated endometrialmaturation but only during the prereceptivesecretory phase and not during the implantation window.Consequently, transfer of a day-3 embryo in such too pre-cociously mature endometrium would not allow the properestablishment of the embryo-endometrium crosstalk; thismight explain why the pregnancy outcome was impairedwhen embryo transfer was performed on day 3 (hCG+5) inpatients with high serum [P] on the day of hCG admin-istration [35]. On the other hand, when embryo transferwas performed on day 5 (hCG+7), no detrimental effect onthe pregnancy outcome was observed [35, 36], particularlyin patients in GnRH antagonist protocols [36]. Anotherstudy reported increased cumulative pregnancy rates inpatients with high serum [P] in GnRH antagonist protocolsfollowing fresh or frozen-thawed embryo transfer [37]. Allthese findings suggest that (i) the periovulatory endometrial
maturation advancement does not necessarily lead to a deficitof endometrial receptivity and (ii) the endometrium canrecover from exposure to supraphysiologic steroid concen-trations [35]. Moreover, as described in Labarta et al. [22],histological dating (Noyes’ criteria, [38]) revealed the absenceof endometrial maturation advancement during the implan-tation window (hCG+7) in patients under COS protocols,regardless of the used GnRH analogues and the serum [P]on the day of hCG administration. In addition, prematureelevation of serum [P] on the day of hCG administrationduring COS did not impact the pregnancy rate in oocytedonation programmes, suggesting a nondeleterious effect ofpremature [P] rise on endometrial receptivity [39, 40].
The mechanisms explaining serum [P] elevation at thetime of hCG administration remain unclear. However, thereare accumulating data suggesting that the main factorsassociated with increased risk of progesterone rise duringCOS cycles are ovarian parameters, including the total FSHdose, the intensity of the ovarian response, and excessnumber of follicles or oocytes [41]. Such parameters werenot significantly different in our two groups of patientsprobably due to the small patients’ number (Table 1). Inaddition, the deleterious effect of premature progesterone riseis probably not due to an impact on endometrial receptivity orovarian parameters but rather to a desynchronized dialoguebetween embryo and endometrium. This hypothesis shouldbe confirmed.
BioMed Research International 9
In conclusion, the gene expression profiles of the endome-trial shift from the prereceptive to the receptive secre-tory stage are altered in patients with high serum [P] onthe day of hCG administration in comparison to patientswith normal [P]. This alteration suggests an acceleration ofendometrial maturation during the periovulatory phase thatshould desynchronize the embryo-endometrium dialogue.On the other hand, endometrial advancement seems todecrease during the perireceptive phase and it does not affectendometrial receptivity.
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper.
Acknowledgments
The authors thank the direction of the University Hospitalof Montpellier and Institute for Research in Biotherapy(IRB) for support and the ART team for their assistanceduring this study. 85% of the specific genes in Figure 4 formpart of protected information under Patent Application no.PCT/EP2011/058757.
References
[1] W. Schoolcraft, E. Sinton, T. Schlenker, D. Huynh, F. Hamilton,and D. R. Meldrum, “Lower pregnancy rate with prema-ture luteinization during pituitary suppression with leuprolideacetate,” Fertility and Sterility, vol. 55, no. 3, pp. 563–566, 1991.
[2] K. M. Silverberg, W. N. Burns, D. L. Olive, R. M. Riehl, andR. S. Schenken, “Serum progesterone levels predict successof in vitro fertilization/embryo transfer in patients stimulatedwith leuprolide acetate and humanmenopausal gonadotropins,”Journal of Clinical Endocrinology and Metabolism, vol. 73, no. 4,pp. 797–803, 1991.
[3] Y. Mio, A. Sekijima, T. Iwabe, Y. Onohara, T. Harada, andN. Terakawa, “Subtle rise in serum progesterone during thefollicular phase as a predictor of the outcome of in vitrofertilization,” Fertility and Sterility, vol. 58, no. 1, pp. 159–166,1992.
[4] A. Shulman, Y. Ghetler, Y. Beyth, and I. Ben-Nun, “Thesignificance of an early (premature) rise of plasma progesteronein in vitro fertilization cycles induced by a “long protocol”of gonadotropin releasing hormone analogue and humanmenopausal gonadotropins,” Journal of Assisted Reproductionand Genetics, vol. 13, no. 3, pp. 207–211, 1996.
[5] E. Bosch, I. Valencia, E. Escudero et al., “Premature luteiniza-tion during gonadotropin-releasing hormone antagonist cyclesand its relationship with in vitro fertilization outcome,” Fertilityand Sterility, vol. 80, no. 6, pp. 1444–1449, 2003.
[6] E. Bosch, E. Labarta, J. Crespo et al., “Circulating progesteronelevels and ongoing pregnancy rates in controlled ovarian stim-ulation cycles for in vitro fertilization: analysis of over 4000cycles,”HumanReproduction, vol. 25, no. 8, pp. 2092–2100, 2010.
[7] A. N. Andersen, P. Devroey, and J.-C. Arce, “Clinical outcomefollowing stimulationwith highly purified hMGor recombinantFSH in patients undergoing IVF: a randomized assessor-blind
controlled trial,” Human Reproduction, vol. 21, no. 12, pp. 3217–3227, 2006.
[8] B. Xu, Z. Li, H. Zhang et al., “Serum progesterone level effectson the outcome of in vitro fertilization in patients with differentovarian response: an analysis of more than 10,000 cycles,”Fertility and Sterility, vol. 97, pp. 1321–1327.e4, 2012.
[9] E. M. Kolibianakis, C. A. Venetis, J. Bontis, and B. C. Tarlatzis,“Significantly lower pregnancy rates in the presence of pro-gesterone elevation in patients treated with GnRH antagonistsand gonadotrophins: a systematic review and meta-analysis,”Current Pharmaceutical Biotechnology, vol. 13, no. 3, pp. 464–470, 2012.
[10] N. Doldi, E. Marsiglio, A. Destefani, A. Gessi, G. Merati, andA. Ferrari, “Elevated serum progesterone on the day of HCGadministration in IVF is associatedwith a higher pregnancy ratein polycystic ovary syndrome,”HumanReproduction, vol. 14, no.3, pp. 601–605, 1999.
[11] M. C. Edelstein, H. J. Seltman, B. J. Cox, S. M. Robinson, R.A. Shaw, and S. J. Muasher, “Progesterone levels on the day ofhuman chorionic gonadotropin administration in cycles withgonadotropin-releasing hormone agonist suppression are notpredictive of pregnancy outcome,” Fertility and Sterility, vol. 54,no. 5, pp. 853–857, 1990.
[12] C. R. Givens, E. D. Schriock, P. V. Dandekar, and M. C. Martin,“Elevated serum progesterone levels on the day of humanchorionic gonadotropin administration do not predict outcomein assisted reproduction cycles,” Fertility and Sterility, vol. 62,no. 5, pp. 1011–1017, 1994.
[13] M. Bustillo, J. J. Stern, and C. B. Coulam, “Serum progesteroneat the time of human chorionic gonadotrophin does not predictpregnancy in in-vitro fertilization and embryo transfer,”HumanReproduction, vol. 10, no. 11, pp. 2862–2867, 1995.
[14] M. I. Abuzeid and M. A. Sasy, “Elevated progesterone levelsin the late follicular phase do not predict success of in vitrofertilization-embryo transfer,” Fertility and Sterility, vol. 65, no.5, pp. 981–985, 1996.
[15] G. E. Hofmann, J. Khoury, C. A. Johnson, J. Thie, and J.Scott R.T., “Premature luteinization during controlled ovarianhyperstimulation for in vitro fertilization-embryo transfer hasno impact on pregnancy outcome,” Fertility and Sterility, vol.66, no. 6, pp. 980–986, 1996.
[16] F. Ubaldi, C. Albano,M. Peukert et al., “Subtle progesterone riseafter the administration of the gonadotrophin-releasing hor-mone antagonist Cetrorelix in intracytoplasmic sperm injectioncycles,” Human Reproduction, vol. 11, no. 7, pp. 1405–1407, 1996.
[17] D. V. Moffitt, J. T. Queenan Jr., B. S. Ruth Shaw, and S. J.Muasher, “Progesterone levels on the day of human chorionicgonadotropin do not predict pregnancy outcome from thetransfer of fresh or cryopreserved embryos from the samecohort,” Fertility and Sterility, vol. 67, no. 2, pp. 296–301, 1997.
[18] B. Urman, C. Alatas, S. Aksoy, R. Mercan, A. Isiklar, andB. Balaban, “Elevated serum progesterone level on the dayof human chorionic gonadotropin administration does notadversely affect implantation rates after intracytoplasmic sperminjection and embryo transfer,” Fertility and Sterility, vol. 72, no.6, pp. 975–979, 1999.
[19] F. Martınez, B. Coroleu, E. Clua et al., “Serum progesteroneconcentrations on the day of HCG administration cannotpredict pregnancy in assisted reproduction cycles,”ReproductiveBioMedicine Online, vol. 8, no. 2, pp. 183–190, 2004.
[20] C. A. Venetis, E. M. Kolibianakis, E. Papanikolaou, J. Bontis, P.Devroey, and B. C. Tarlatzis, “Is progesterone elevation on the
10 BioMed Research International
day of human chorionic gonadotrophin administration associ-ated with the probability of pregnancy in in vitro fertilization?A systematic review and meta-analysis,” Human ReproductionUpdate, vol. 13, no. 4, pp. 343–355, 2007.
[21] H. A. Saleh, M. S. E.-A. Omran, and M. Draz, “Does subtleprogesterone rise on the day of HCG affect pregnancy rate inlong agonist ICSI cycles?” Journal of Assisted Reproduction andGenetics, vol. 26, no. 5, pp. 239–242, 2009.
[22] E. Labarta, J. A. MartInez-Conejero, P. Alama et al., “Endome-trial receptivity is affected in women with high circulating pro-gesterone levels at the end of the follicular phase: a functionalgenomics analysis,” Human Reproduction, vol. 26, no. 7, pp.1813–1825, 2011.
[23] R. Li, J. Qiao, L. Wang et al., “MicroRNA array and microarrayevaluation of endometrial receptivity in patients with highserum progesterone levels on the day of hCG administration,”Reproductive Biology and Endocrinology, vol. 9, article 29, 2011.
[24] E. Kolibianakis, C. Bourgain, C. Albano et al., “Effect of ovarianstimulation with recombinant follicle-stimulating hormone,gonadotropin releasing hormone antagonists, and humanchorionic gonadotropin on endometrial maturation on the dayof oocyte pick-up,” Fertility and Sterility, vol. 78, no. 5, pp. 1025–1029, 2002.
[25] C. Simon, J. Oberye, J. Bellver et al., “Similar endometrialdevelopment in oocyte donors treated with either high- orstandard-dose GnRH antagonist compared to treatment with aGnRH agonist or in natural cycles,” Human Reproduction, vol.20, no. 12, pp. 3318–3327, 2005.
[26] I. Van Vaerenbergh, L. Van Lommel, V. Ghislain et al.,“In GnRH antagonist/rec-FSH stimulated cycles, advancedendometrialmaturation on the day of oocyte retrieval correlateswith altered gene expression,”Human Reproduction, vol. 24, no.5, pp. 1085–1091, 2009.
[27] I. Van Vaerenbergh, H. M. Fatemi, C. Blockeel et al., “Pro-gesterone rise on HCG day in GnRH antagonist/rFSH stimu-lated cycles affects endometrial gene expression,” ReproductiveBioMedicine Online, vol. 22, no. 3, pp. 263–271, 2011.
[28] D.Haouzi, K.Mahmoud,M. Fourar et al., “Identification of newbiomarkers of human endometrial receptivity in the naturalcycle,” Human Reproduction, vol. 24, no. 1, pp. 198–205, 2009.
[29] D. Haouzi, S. Assou, K. Mahmoud et al., “Gene expressionprofile of human endometrial receptivity: comparison betweennatural and stimulated cycles for the same patients,” HumanReproduction, vol. 24, no. 6, pp. 1436–1445, 2009.
[30] V. G. Tusher, R. Tibshirani, and G. Chu, “Significance analysisof microarrays applied to the ionizing radiation response,”Proceedings of the National Academy of Sciences of the UnitedStates of America, vol. 98, no. 9, pp. 5116–5121, 2001.
[31] C.-C. Chang, Y.-Y. Hsieh, K.-H. Hsu, and C.-S. Lin, “Effects of 𝛼and 𝛽 recombinant FSH (Gonal-F, Puregon) and progesteroneupon human endometrial cell proliferation in-vitro: a prelimi-nary study,” Gynecological Endocrinology, vol. 27, no. 2, pp. 110–116, 2011.
[32] D. Haouzi, H. Dechaud, S. Assou, J. De Vos, and S. Hamamah,“Insights into human endometrial receptivity from transcrip-tomic and proteomic data,” Reproductive BioMedicine Online,vol. 24, no. 1, pp. 23–34, 2012.
[33] J. A. Horcajadas, A. Pellicer, and C. Simon, “Wide genomicanalysis of human endometrial receptivity: new times, newopportunities,” Human Reproduction Update, vol. 13, no. 1, pp.77–86, 2007.
[34] P. Dıaz-Gimeno, J. A.Horcajadas, J. A.Martınez-Conejero et al.,“A genomic diagnostic tool for human endometrial receptivitybased on the transcriptomic signature,” Fertility and Sterility,vol. 95, no. 1, pp. 50–60.e15, 2011.
[35] E. G. Papanikolaou, E. M. Kolibianakis, C. Pozzobon et al.,“Progesterone rise on the day of human chorionic gonadotropinadministration impairs pregnancy outcome in day 3 single-embryo transfer, while has no effect on day 5 single blastocysttransfer,” Fertility and Sterility, vol. 91, no. 3, pp. 949–952, 2009.
[36] R. Ochsenkuhn, A. Arzberger, V. von Schonfeldt et al., “Subtleprogesterone rise on the day of human chorionic gonadotropinadministration is associated with lower live birth rates inwomen undergoing assisted reproductive technology: a retro-spective study with 2,555 fresh embryo transfers,” Fertility andSterility, vol. 98, pp. 347–354, 2012.
[37] H. M. Fatemi, K. Doody, G. Griesinger, H. Witjes, and B. Man-naerts, “High ovarian response does not jeopardize ongoingpregnancy rates and increases cumulative pregnancy rates in aGnRH-antagonist protocol,” Human Reproduction, vol. 28, no.2, pp. 442–452, 2012.
[38] R. W. Noyes, A. T. Hertig, and J. Rock, “Dating the endometrialbiopsy,” Fertility and Sterility, vol. 1, pp. 3–25, 2012.
[39] R. S. Legro, B. A. Ary, R. J. Paulson, F. Z. Stanczyk, and M. V.Sauer, “Premature luteinization as detected by elevated serumprogesterone is associated with a higher pregnancy rate indonor oocyte in-vitro fertilization,” Human Reproduction, vol.8, no. 9, pp. 1506–1511, 1993.
[40] M. A. B. Melo, M. Meseguer, N. Garrido, E. Bosch, A. Pellicer,and J. Remohı, “The significance of premature luteinization inan oocyte-donation programme,”Human Reproduction, vol. 21,no. 6, pp. 1503–1507, 2006.
[41] J. N. Hugues, “Impact of “LH activity” supplementation onserum progesterone levels during controlled ovarian stimula-tion: a systematic review,” Human Reproduction, vol. 27, no. 1,pp. 232–243, 2012.
